1. Field of the Invention
The present invention relates to a novel polyhydroxyalkanoate (PHA), a method for production of such PHA and microorganisms for use in the same.
2. Related Background Art
Synthetic polymers derived from petroleum have been used as plastics etc. for a long time. Recently, the treatment of the used plastics has become one of serious social problems. These synthetic polymers have advantages of hard-to-decompose have been used in the place of metal or glass materials. On mass consumption and mass disposal, however, this feature of hard-to-decompose makes them accumulated in waste-disposal facilities, or when they are burned, it causes increased carbon dioxide exhaust, and harmful substances such as dioxin and endocrine-disruptors may be generated to cause environmental pollution. On the other hand, polyhydroxyalkanoates (PHAs) produced by microorganisms (hereinafter referred to as xe2x80x9cmicrobial polyesterxe2x80x9d) represented by poly-3-hydroxy butyric acid (PHB) can be used as the conventional plastics to make various kinds of products with melting processes etd., and can be decomposed by organisms unlike oil-derived synthetic polymers. Therefore, the microbial polyester is bio-decomposed and thus incorporated in the natural material cycle when discarded, and would not remain in the natural environment to cause pollution unlike many conventional synthetic polymer compounds. Furthermore, since the microbial polyesters do not require incineration processes, they are also effective in terms of prevention of air pollution and global warming. Thus, they can be used as a plastic enabling environmental integrity. In addition, the application of the microbial polyesters to medical soft members is under consideration (Japanese Patent Application Laid-Open No. 5-159, Japanese Patent Application Laid-Open No. 6-169980, Japanese Patent Application Laid-Open No. 6-169988, Japanese Patent Application Laid-Open No. 6-225921 and the like).
Heretofore, various bacteria have been reported to produce and accumulate PHB or copolymers of other hydroxyalkanoic acids in the cells (xe2x80x9cBiodegradable Plastics Handbookxe2x80x9d, edited by Biodegradable Plastics Society, issued by NTS Co. Ltd., P178-197, (1995)). It is known that such microbial PHAs may have a variety of compositions and structures depending on types of the producing microorganisms, the composition of culture media, culture conditions and the like, and up to now, studies regarding the control of these compositions and structures have been carried out to improve the properties of PHA.
For example, Alcaligenes eutropus H16 (ATCC No. 17699) and its mutant strains reportedly produce copolymers of 3-hydroxy butyric acid (3HB) and 3-hydroxy valeric acid (3HV) at a variety of composition ratios according to the carbon source in culture (Japanese Patent Publication No. 6-15604, Japanese Patent Publication No. 7-14352, Japanese Patent Publication No. 8-19227 and the like).
Japanese Patent Application Laid-Open No. 5-74492 discloses a method in which the copolymer of 3HB and 3HV is produced by bringing Methylobacterium sp., Paracoccus sp., Alcalugenes sp. or Pseudomonas sp. into contact primary alcohol having 3 to 7 carbons.
Japanese Patent Application Laid-Open No. 5-93049 and Japanese Patent Application Laid-Open No. 7-265065 disclose that two-component copolymers of 3HB and 3-hydroxy hexanoic acid (3HHx) are produced by culturing Aeromonas caviae using oleic acid or olive oil as a carbon source.
Japanese Patent Application Laid-Open No. 9-191893 discloses that Comamonas acidovorans IFO 13852 produces polyester having 3HB and 4-hydroxy butyric acid as monomer units in culture with gluconic acid and 1,4-butandiol as a carbon source.
Also, in recent years, active researches about PHA composed of 3-hydroxyalkanoate (3HA) of medium-chain-length (abbreviated to mcl) having up to about 12 carbons. Synthetic routes can be classified broadly into two types, and their specific examples will be shown in (1) and (2) below.
(1) Synthesis Using xcex2-Oxidation
Japanese Patent No. 2642937 discloses that PHA having monomer units of 3-hydroxyalkanoate having 6 to 12 carbons is produced by providing as a carbon source aliphatic hydrocarbon to Pseudomonas oleovorans ATCC 29347. Furthermore, it is reported in Appl. Environ. Microbiol, 58(2), 746 (1992) that Pseudomonas resinovorans produces polyester having 3-hydroxybutyric acid, 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid and 3-hydroxydecanoic acid at a ratio of 1:15:75:9 as monomer units, using octanoic acid as a single carbon source, and also produces polyester having 3-hydroxybutyric acid, 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid and 3-hydroxydecanoic acid (quantitative ratio of 8:62:23:7) as units, using hexanoic acid as a single carbon source. Herein, it is assumed that 3HA monomer units having longer chain length than that of the starting fatty acid are made by way of fatty acid synthetic route that will be described next in (2).
(2) Synthesis Using Fatty Acid Synthetic Route
It is reported in Int. J. Biol. Macromol., 16(3), 119 (1994) that Pseudomonas sp. 61-3 strain produces polyester made of 3-hydroxyalkanoic acids such as 3-hydroxybutyric acid, 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, 3-hydroxydecanoic acid and 3-hydroxydodecanoic acid and 3-hydroxyalkenoic acids such as 3-hydroxy-5-cis-decenoic acid, 3-hydroxy-5-cis-dodecenoic acid, using sodium gluconate as a single carbon source.
By the way, the biosynthesis of PHA is usually carried out by a PHA synthase using as a substrate xe2x80x9cD-3-hydroxyacyl-CoAxe2x80x9d occurring as an intermediate of a variety of metabolic pathways in the cell.
Herein, xe2x80x9cCoAxe2x80x9d means a xe2x80x9ccoenzyme Axe2x80x9d. And, as described in the prior art of the above (1), the biosynthesis of PHA is carried out with xe2x80x9cD-3-hydroxyacyl-CoAxe2x80x9d occurring in the xe2x80x9cxcex2 oxidation cyclexe2x80x9d being a starting substance in the case where fatty acids such as octanoic acid and nonanoic acid are used as carbon sources.
Reactions through which PHA is synthesized by way of the xe2x80x9cxcex2 oxidation cyclexe2x80x9d will be shown below. 
On the other hand, as described in the prior art of the above (2), in the case where the PHA is biosynthesized using saccharides such as glucose and the like, the biosynthesis is carried out with xe2x80x9cD-3-hydroxyacyl-CoAxe2x80x9d converted from xe2x80x9cD-3-hydroxyacyl-ACPxe2x80x9d occurring in the xe2x80x9cfatty acid synthesis pathwayxe2x80x9d being a starting substance.
Herein, xe2x80x9cACPxe2x80x9d means a xe2x80x9cacyl carrier proteinxe2x80x9d.
By the way, as described previously, the PHA synthesized in both (1) and (2) described above is PHA constituted by monomer units having alkyl groups in side chains. However, if a wider range of application of the microbial PHA like this, for example an application as a functional polymer is considered, it is expected that PHA having various substituents (for example phenyl groups) introduced in the side chain is significantly useful. With respect to the synthesis of such PHA, for the synthesis using xcex2 oxidation, a report regarding PHA having the aryl group and the like in the side chain can be found in, for example, Macromolecules, 24, p5256-5260 (1991). Specifically, it is reported that Pseudomonas oleovorans produces polyester having 3-hydroxy valeric acid, 3-hydroxyheptanoic acid, 3-hydroxynonanoic acid, 3-hydroxyundecanoic acid and 3-hydroxy-5-phenyl valeric acid (quantitative ratio of 0.6:16.0:41.1:1.7:40.6) as units in the amount of 160 mg for 1 L of culture solution (ratio in dry weight to the cell mass is 31.6%), using 5-phenylvaleric acid and nonanoic acid (mole ratio of 2:1, total concentration of 10 mmol/L) as a medium, and also produces polyester having 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, 3-hydroxydecanoic acid and 3-hydroxy-5-phenyl valeric acid (quantitative ratio of 7.3:64.5:3.9:24.3) as units in the amount of 200 mg for 1 L of culture solution (ratio in dry weight to the cell mass is 39.2%), using 5-phenyl valeric acid and octanoic acid (mole ratio of 1:1, total concentration of 10 mmol/L). It is conceivable that the PHA in this report is principally synthesized by way of the xcex2 oxidation pathway due to that fact that nonanoic acid and octanoic acid are used.
As described above, in the microbial PHA, those with various kinds of compositions/structures are obtained by changing the type of microorganisms for use in its production, culture medium compositions, culture conditions, but if considering the application of the microbial PHA as plastics, they could not be sufficient yet in terms of properties. In order to further expand the range of the microbial PHA utility, it is important that the improvement of its properties are more widely considered, and for this purpose, the development and the search of the PHA containing monomer units of further diverse structures, its manufacturing processes and microorganisms enabling desired PHA to be produced efficiently are essential.
On the other hand, the PHA of a type having substituents introduced in the side chain as described previously is selected in accordance with the property for which the introduced substituent is desired, thereby making it possible to expect its development as a xe2x80x9cfunctional polymerxe2x80x9d having very useful functions and properties resulting from the property and the like of the introduced substituent, and the development and the search of excellent PHA allowing such functionality and the biodegradability to be compatible with each other, its manufacturing processes and microorganisms enabling desired PHA to be produced efficiently are also important challenges.
Another example of such PHA having substituents introduced in the side chain includes PHA having the above described phenyl groups, and further phenoxy groups in the side chain.
For another example of phenyl group, it is reported in Macromolecules, 29, 1762-1766 (1996) that Pseudomonas oleovorans produces PHA including 3-hydroxy-5-(4-toryl)valeric acid as a monomer unit through the culture in a culture medium including 5-(4-toryl)valeric acid (5-(4-methylphenyl)valeric acid) as a substrate.
Furthermore, it is reported in Macromolecules, 32, 2889-2895 (1999) that Pseudomonas oleovorans produces PHA including 3-hydroxy-5-(2,4-dinitrophenyl)valeric acid and 3-hydroxy-5-(4-nitrophenyl)valeric acid as monomer units through the culture in a culture medium including 5-(2,4-dinitrophenyl)valeric acid and nonanoic acid as a substrate.
Also, for an example of the phenoxy group, it is reported in Macromol. Chem. Phys., 195, 1665-1672 (1994) that Pseudomonas oleovorans produces PHA including 3-hydroxy-5-phenoxy valeric acid and 3-hydroxy-9-phenoxynonanoic acid as units from 11-phenoxyundecanoic acid.
Also, it is reported in Macromolecules, 29, 3432-3435 (1996) that Pseudomonas oleovorans is used to produce PHA including 3-hydroxy-4-phenoxybutyric acid and 3-hydroxy-6-phenoxyhexanoic acid as units from 6-phenoxyhexanoic acid, PHA including 3-hydroxy-4-phenoxybutyric acid, 3-hydroxy-6-phenoxyhexanoic acid and 3-hydroxy-8-phenoxyoctanoic acid as units from 8-phenoxyoctanoic acid, and PHA including 3-hydroxy-5-phenoxyvaleric acid and 3-hydroxy-7-phenoxyheptanoic acid as units from 11-phenoxyundecanoic acid. Excerpts of yields of polymers from this report are shown in Table 1.
Furthermore, in Can. J. Microbiol., 41, 32-43 (1995), PHA including 3-hydroxy-p-cyanophenoxyhexanoic acid or 3-hydroxy-p-nitrophenoxyhexanoic acid as a monomer unit is successfully produced with octanoic acid and p-cyanophenoxyhexanoic acid or p-nitrophenoxyhexanoic acid being a substrate, using Pseudomonas oleovorans ATCC 29347 and Pseudomonas putida KT 2442.
In Japanese Patent No. 2989175, a homopolymer constituted by 3-hydroxy-5-(monofluorophenoxy)pentanoate(3H5(MFP)P) units or 3-hydroxy-5-(difluorophenoxy)pentanoate(3H5(DFP)P) units and a copolymer containing at least 3H5(MFP)P units or 3H5(DFP)P units; Pseudomonas putida for synthesizing these polymers; and a method of producing the aforesaid polymers using Pseudomonas species are described.
These productions are carried out through xe2x80x9ctwo-stage culturexe2x80x9d as described below.
Time of culture: First stage, 24 hours ; Second stage, 96 hours
A substrate and a resulting polymer at each stage will be shown below.
(1) Resulting polymer: 3H5 (MFP) P homopolymer
Substrate at the first stage: Citric acid, Yeast extract
Substrate at the second stage: Monofluorophenoxyundecanoic acid
(2) Resulting polymer: 3H5 (DFP) P homopolymer
Substrate at the first stage: Citric acid, Yeast extract
Substrate at the second stage: Difluorophenoxyundecanoic acid
(3) Resulting polymer: 3H5 (MFP) P copolymer
Substrate at the first stage: Octanoic acid or Nonanoic acid, Yeast extract
Substrate at the second stage: Monofluorophenoxyundecanoic acid
(4) Resulting polymer: 3H5 (DFP) P copolymer
Substrate at the first stage: Octanoic acid or Nonanoic acid, Yeast extract
Substrate at the second stage: Difluorophenoxyundecanoic acid
As its effect, a medium-chain-length fatty acid having substituents may be materialized to synthesize a polymer having phenoxy groups with ends of the side chain replaced by one to two fluorine atoms, and stereoregularity and water repellency can be provided while maintaining a high melting point and good processability.
Also, PHA including cyclohexyl groups in monomer units is expected to show polymeric properties different from those of PHA including normal aliphatic hydroxyalkanoic acid as a unit, and an example of production using Pseudomonas oleovorans has been reported (Macromolecules, 30, 1611-1615 (1997)).
According to this report, when Pseudomonas oleovorans was cultured in a culture medium where nonanoic acid (hereinafter described as NA) and cyclohexylbutyric acid (hereinafter described as CHBA) or cyclohexyl valeric acid (hereinafter described as CHVA) coexisted, PHA including units containing cyclohexyl groups and units originating from nonanoic acid were obtained (each ratio unknown)
About the yields, it is reported that quantitative ratios of CHBA and NA are varied with substrate concentration total of 20 mmol/L and results as shown in Table 2 were obtained.
However, in this example, the yield of polymers per culture solution is not sufficient, and the obtained PHA itself has aliphatic hydroxyalkanoic acid coexist in its monomer unit.
In this way, in the case where PHA with a variety of substituents introduced in the side chain is produced, as seen in the reported example of Pseudomonas oleovorans described previously and the like, a method is used in which alkanoate having a substituent to be introduced is used not only as a stock for the polymer but also as a carbon source for growth.
However, for the method in which alkanoate having a substituent to be introduced is used not only as a stock for the polymer but also as a carbon source for growth, the supply of an energy source based on the production of the acetyl-CoA by xcex2 oxidation from such alkanoate is expected, and in this method, only a substrate having a certain degree of chain length is capable of producing acetyl-CoA by xcex2 oxidation, thus limiting alkanoate that can be used as a substrate of PHA, which is a major problem. Also, generally, since substrates with the chain length decreased by two methylene chains an after another are newly produced by the xcex2 oxidation, and these are captured as monomer units of PHA, the PHA that is synthesized is often a copolymer constituted by monomer units that are different in the chain length by two methylene chains one after another. In the reported example described above, a copolymer constituted by three types of monomer units, that is 3-hydroxy-8-phenoxyoctanoic acid originating from 8-phenoxyoctanoic acid which is a substrate, 3-hydroxy-6-phenoxyhexanoic acid and 3-hydroxy-4-phenoxybutyric acid which are by-products originating from metabolites is produced. In this respect, if PHA constituted by single monomer units is to be obtained, it is quite difficult to use this method. Furthermore, for a method premised on the supply of an energy source based on the production of acetyl-CoA by the xcex2 oxidation, the growth of microorganisms is slow and the synthesis of PHA requires lots of time, and the yield of the synthesized PHA is often low, which is also a major problem.
For this reason, a method in which, in addition to the alkanoate having substituents to be introduced, microorganisms are cultured in the culture medium in which fatty acids of medium-chain-length and the like such as octanoic acid and nonanoic acid as the carbon source for growth, followed by extracting PHA is considered to be effective and is generally used.
However, according to the study by the inventors, the PHA synthesized by way of the xcex2 oxidation pathway using fatty acids of medium-chain-length such as octanoic acid and nonanoic acid as the carbon source for growth has poor purity, and 50% or more of the polymers are made of mcl-3HA monomer units originating from the carbon source (for example, 3-hydroxyoctanoic acid, 3-hydroxynonanoic acid and the like). These mcl-3HA units make polymers adhesive at room temperature when they are sole components, and if they coexist in the PHA of the present invention in large quantity, the glass transition temperature (Tg) of the polymer is significantly lowered. Thus, to obtain hard polymers at room temperature, the coexistence of mcl-3HA monomer units is not desired. Also, it is known that such a hetero-side chain structure interferes intra-molecular or inter-molecular interaction originating from the side chain structure, and has significant influence on crystallinity and orientation. For achieving the improvement of polymer properties and the addition of functionality, the coexistence of these mcl-3HA monomer units raises a major problem. Means for solving this problem includes providing a refinement process to separate/remove xe2x80x9cundesiredxe2x80x9d monomer units such as mcl-3HA monomer units originating from the carbon source for growth, in order to acquire PHA constituted by monomer units having only specified substituents. However, the problem is that operations are complicated and a significant decrease in the yield can not be avoided. A more serious problem is that if desired monomer units and undesired monomer units form a copolymer, it is quite difficult to remove only undesired monomer units. Particularly, in the case where the purpose is to synthesize PHA including monomer units having groups obtained from unsaturated hydrocarbons, ester groups, aryl groups, cyan groups, nitro groups, groups obtained from halogenated hydrocarbons, groups having epoxide and the like introduced therein as side chain structures, the mcl-3HA monomer unit often forms a copolymer with a desired monomer unit, and it is extremely difficult to remove the mcl-3HA monomer unit after PHA is synthesized.
The present invention solves the above described problems, and provides PHA including monomer units of diverse structures having substituents in the side chain, which is useful as device materials, medical materials and the like, and a method of producing such PHA using microorganisms, and particularly a production method in which the coexistence of undesired monomer units is reduced, desired PHA can be obtained in high purity and also in high yields. The present invention is also intended to provide strains enabling such PHA to be synthesized in high purity and with efficiency.
According to one aspect of the present invention, there is provided a polyhydroxyalkanoate having a monomer unit composition represented by a general formula (1):
AmB(1xe2x88x92m)xe2x80x83xe2x80x83(1)
wherein A is represented by General Formula (2), B is at least one selected from the group consisting of monomer units represented by General Formula (3) or (4), and m has a value of 0.01 or larger and smaller than 1: 
xe2x80x83wherein
n has a value of 0 to 10, k has a value of 3 or 5, and
R is at least one group selected from the group consisting of groups represented by General Formulae (5) to (7): 
xe2x80x83in Formula (5)
R1 is a group selected from the group consisting of a hydrogen atom (H), halogen atoms, xe2x80x94CN, xe2x80x94NO2, xe2x80x94CF3, xe2x80x94C2F5 and xe2x80x94C3F7; and q is an integer selected from 1 to 8;
xe2x80x83in Formula (6)
R2 is a group selected from the group consisting of a hydrogen atom (H), halogen atoms, xe2x80x94CN, xe2x80x94NO2, xe2x80x94CF3, xe2x80x94C2F5 and xe2x80x94C3F7; and r is an integer selected from 1 to 8;
xe2x80x83in Formula (7)
R3 is a group selected from the group consisting of a hydrogen atom (H), halogen atoms, xe2x80x94CN, xe2x80x94NO2, xe2x80x94CF3, xe2x80x94C2F5 and xe2x80x94C3F7; and s is an integer selected from 1 to 8;
provided that following R are excluded from the choice:
when selecting one type of group as R in the general formula (2):
groups of Formula (5) in which R1 is H and q=2, R1 is H and q=3, and R1 is xe2x80x94NO2 and q=2;
groups of Formula (6) in which R2 is a halogen atom and r=2, provided that two components are selected as B from General Formula (3) or (4), R2 is xe2x80x94CN and r=3, and R2 is xe2x80x94NO2 and r=3; and the groups of Formula (7) in which R3 is H and s=1, and R3 is H and s=2; and
when selecting two types of groups as R in General Formula (2), the combinations of two types of groups of Formula (6) in which R2 is a halogen atom and r=2, and R2 is a halogen atom and r=4, provided that one component is selected as B from General Formula (3) or (4).
According to one aspect of the present invention, there is provided 5-(4-trifluoromethylphenyl)valeric acid of Formula (21). 
According to another aspect of the present invention, there is provided a method of producing a polyhydroxyalkanoate, comprising a step of culturing a microorganism capable of synthesizing a polyhydroxyalkanoate of which monomer unit is represented by Formula (1) from an alkanoate in a medium containing the alkanoate:
AmB(1xe2x88x92m)xe2x80x83xe2x80x83(1)
wherein A is represented by General Formula (2), B is at least one selected from the group consisting of monomer units represented by General Formula (3) or (4), and m is 0.01 or larger and smaller than 1, 
xe2x80x83wherein
n is an integer selected from 0 to 10, k is 3 or 5, and
R is at least one group selected from the group consisting of the groups represented by General Formulae (5) to (7): 
xe2x80x83in Formula (5)
R1 is a group selected from the group consisting of a hydrogen atom (H), halogen atoms, xe2x80x94CN, xe2x80x94NO2, xe2x80x94CF3, xe2x80x94C2F5 and xe2x80x94C3F7; and q is an integer selected from 1 to 8;
xe2x80x83in Formula (6)
R2 is a group selected from the group consisting of a hydrogen atom (H), halogen atoms, xe2x80x94CN, xe2x80x94NO2, xe2x80x94CF3, xe2x80x94C2F5 and xe2x80x94C3F7; and r is an integer selected from 1 to 8;
xe2x80x83in Formula (7)
R3 is a group selected from the group consisting of a hydrogen atom (H), halogen atoms, xe2x80x94CN, xe2x80x94NO2, xe2x80x94CF3, xe2x80x94C2F5 and xe2x80x94C3F7; and s is an integer selected from 1 to 8;
provided that following R are excluded from the choice:
when selecting one type of group as R in General Formula (2):
groups of Formula (5) in which R1 is H and q=2,
R1 is H and q=3, and R1 is xe2x80x94NO2 and q=2; the groups of Formula (6) in which R2 is a halogen atom and r=2, R2 is xe2x80x94CN and r=3, and R2 is xe2x80x94NO2 and r=3; and
groups of Formula (7) in which R3 is H and s=1, and R3 is H and s=2; and
when selecting two types of groups as R in General Formula (2),
groups of Formula (6) in which R2 is a halogen atom and r=2.
According to a further aspect of the present invention, there is provided a process of producing polyhydroxyalkanoate, comprising a step of culturing a microorganism capable of producing the polyhydroxyalkanoate utilizing alkanoate in a medium containing the alkanoate and a saccharide.
According to a further aspect of the present invention, there is provided a process of producing polyhydroxyalkanoate, comprising a step of culturing a microorganism capable of producing a polyhydroxyalkanoate utilizing an alkanoate in a medium containing the alkanoate and a polypeptone.
According to a further aspect of the present invention, there is provided a process of producing polyhydroxyalkanoate comprising a step of culturing a microorganism capable of producing a polyhydroxyalkanoate utilizing an alkanoate in a medium containing the alkanoate and an organic acid participating in TCA cycle.
According to a further aspect of the present invention, there is provided a process of producing polyhydroxyalkanoate, wherein the a microorganism is cultured in at least two steps: one is in a medium containing an alkanoate and a polypeptone and the subsequent one is in a medium containing the alkanoate and pyruvic acid or salt thereof with nitrogen source limitation.
According to a further aspect of the present invention, there is provided Pseudomonas cichorii H45, FERM BP-7374.
According to a further aspect of the present invention, there is provided a novel bacterial strain Pseudomonas cichorii YN2, FERM BP-7375.
According to a further aspect of the present invention, there is provided a novel bacterial strain Pseudomonas putida P91, FERM BP-7373.
According to a further aspect of the present invention, there is provided a novel bacterial strain Pseudomonas jessenii P161, FERM BP-7376.
As already stated above, the present invention provides novel polyhydroxyalkanoates and novel substituted alkanoic acids to be a raw material therefor and novel microorganisms which have ability to produce and accumulate in the cell the novel polyhydroxyalkanoates, and provides methods for producing the polyhydroxyalkanoates using such microorganism. According to them, the polyhydroxyalkanoates useful as functional polymers in which different functional groups are introduced can be manufactured very efficiently and in high purity, therefore it may be expected to be applied to each field such as device and medical materials.